All viruses must take over the host's protein synthesis (translation) machinery. Cellular mRNAs require a 5' cap and poly(A) tail to recruit the ribosome and initiate translation in a regulated manner. Many viral RNAs avoid this control step, and avoid host defenses, by lacking a 5' cap or poly(A) tail. Instead, many viral mRNAs harbor sequences in the untranslated regions (UTRs) that facilitate highly efficient cap-independent translation. Understanding how viruses do this could lead to development of antiviral agents that specifically target unique viral translation mechanisms. This knowledge could also allow exploitation of viruses as gene therapy vectors in humans, or as expression vectors to produce custom pharmaceutical polypeptides in plants. This proposal focuses on the novel cap-independent translation element (BTE) in the 3' UTR of barley yellow dwarf (BYDV) and other viral RNAs that facilitates translation initiation at the 5' end of the RNA. This process requires long-distance base pairing between the 5' and 3' UTRs. Our goal is to determine how the BTE recruits the translational machinery. In Aim I we will determine the sequence and structural requirements of the BTE at high resolution by high volume mutagenesis, and translation in cell-free wheat germ extracts and in plant protoplasts. In Aim II we will dissect the role and structural requirements of translation initiation factors elF4G and elF4E, and possibly other factors that are required for BTE-mediated translation. We will observe binding of mutant factors with the BTE RNA by filter binding, surface plasmon resonance, and RNA footprinting assays. The functions of mutant factors will be discerned by reconstituting factor-depleted cell-free extracts, and by depleting cells of factors via virus-induced gene silencing. In Aim , the mechanism of ribosome entry on the RNA will be investigated by sucrose gradient centrifugation of RNA-ribosome complexes, toeprinting, and other approaches. Throughout the project, the role of the BTE and its interactors in virus replication will be assessed. This research on a model virus and major plant pathogen may contribute to understanding picornaviruses (e.g. polio) that also employ cap-independent translation regulated by interactions between the UTRs, and nidoviruses (e.g. SARS) and flaviviruses (e.g. dengue, West Nile) that regulate gene expression and replication by long-distance RNA base pairing. Finally, the research will provide fundamental insight on eukaryotic translation initiation mechanisms.